Liyanagedera, Sahan B. W. (2022) Optimisation of cell-free transcription/translation systems for phage therapy. PhD thesis, University of Warwick.

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Abstract

The last decade has seen the emergence of multi-drug resistant pathogens as a lead- ing cause of death worldwide, reigniting interest in the field of phage therapy. Modern advances in the genetic engineering of bacteriophages have enabled several useful results including hon strange alterations, constitutively tic growth, and control over phage repli- cation.
However, the slow licensing process of genetically modified organisms clearly inhibits the rapid therapeutic application of novel engineered variants necessary to fight mutant pathogens that emerge throughout the course of a pandemic. As a solution to these problems, this thesis aims to provide generally applicable tools to enable rapid and scal- able engineering of phage-based therapies using cell-free systems. To this end, a novel rapid cell-free phage engineering pipeline, the SpyPhage system, is developed. The system uses a "scaffold" bacteriophage which is engineered to incorporate a SpyTagmoiety on its capsid head to enable rapid post-synthetic modification of their surfaces with SpyCatcher fused therapeutic proteins. As a proof of concept, through CRISPR/Cas-facilitated phage engineering and a novel de novo genome assembly protocol, the SpyTag protein is targeted as a capsid fusion to K1F,a phage targeting the pathogenic strain E. coli K1. A cell-free system is used to achieve the first known demonstration of (1)cell-free assembly and(2) a post-translational decoration of the phage surface with therapeutic fusion proteins. This cell-free phage production pipeline proposed enables the generation of multiple phenotypi- cally distinct phage with a single underlying “scaffold” genotype. These phages could be- come the basis of next-generation phage therapies where the knowledge-based engineering of numerous phage variants would be quickly achievable without the use of live bacteria or the need to repeatedly license novel genetic alterations. The increasing incidence of bacterial infections caused by antibiotic-resistant pathogens worldwide also underlines the need to develop novel diagnostic tools enabling the early initiation of targeted antimicrobial therapy. One promising possibility is to unite the high specificity and sensitivity of phage- based applications with the speed and sensitivity provided by microfluidic devices. As a prerequisite for developing such systems, a simple and efficient method is presented for the directional immobilization of phages on the surface of Polydimethylsiloxane (PDMS) chips, a material commonly used for building such devices. This work utilises the covalent interaction between two proteins: SpyTag, expressed by the phage, and BslA-Spycatcher fusion protein, purified and immobilized on PDMS surfaces. The use of SpyTagged phage enables the faciledirectional immobilisation of phage on to PDMS-based microfluidicde- vices. This technique serves to illustrate a generally applicable solution to develop the next generation of phage-based bio-sensors.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QH Natural history > QH426 Genetics Q Science > QR Microbiology Q Science > QR Microbiology > QR355 Virology T Technology > TP Chemical technology
Library of Congress Subject Headings (LCSH):	Bacteriophages -- Genetic engineering, Bacteriophages -- Therapeutic use, Genetic transcription, Biosensors, Self-assembly (Chemistry), CRISPR (Genetics)
Official Date:	June 2022
Dates:	Date Event June 2022 UNSPECIFIED
Institution:	University of Warwick
Theses Department:	School of Engineering
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Kulkarni, Vishwesh
Format of File:	pdf
Extent:	xv, 168 pages : illustrations, chart
Language:	eng

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